Journal: JCI Insight

Article Title: The homozygous CX3CR1-M280 mutation impairs human monocyte survival

doi: 10.1172/jci.insight.95417

Figure Lengend Snippet: Shown are percent of dead monocytes elicited by serum starvation in the presence or absence of 100 nM of CX3CL1 after exposure to 50 μM of the PI3K inhibitor LY294002 (A, n = 5) or 20 μM of the MEK inhibitor PD98059 (B, n = 6), or 100 nM of the PI3K inhibitor wortmannin (C, n = 6) or 20 μM of the MEK inhibitor U0126 (D, n = 5). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons test. Data represent the mean ± SEM.

Article Snippet: Monocytes (1 x 10 5 ) were pretreated for 20 minutes at 37 o C with either the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 (50 μM; Tocris Bioscience, catalog 1130) or the PI3K inhibitor wortmannin (100 nM; Tocris Bioscience, catalog 1232), or the MEK inhibitor PD98059 (20 μM; Tocris Bioscience, catalog 1213) or the MEK inhibitor U0126 (20 μM; Tocris Bioscience, catalog 1144) in serum free medium.

Techniques:

Journal: Microorganisms

Article Title: Three Lipid Emulsions Reduce Staphylococcus aureus -Stimulated Phagocytosis in Mouse RAW264.7 Cells

doi: 10.3390/microorganisms9122479

Figure Lengend Snippet: The activation of PI3K and JNK is involved in the lipid emulsion-caused reduction in phagocytosis. ( a ) The increase of p-JNK in S. aureus -infected RAW264.7 cells during the indicated time points was detected by western bolt analysis. ( b ) The phosphorylation of AKT, JNK, and ERK in S. aureus -infected RAW264.7 cells with or without lipid emulsion treatment was determined by western blot analysis. ( c ) The inhibitors’ effects on the phagocytosis of RAW264.7 cells were quantified by pHrodo™ Green S. aureus Bioparticles ® conjugate in the absence (positive control) or presence of indicated reagents. The reagents included the inhibitors of PI3K (LY294002), JNK (SP600125), ERK (PD98059), and phagocytosis (cytochalasin D). ( d ) Image of phagocytosis in live cells. RAW264.7 cells were treated with and without inhibitors prior to and during phagocytosis. The nuclei of cells were stained with Hoechst 33342 (blue). The location of the lysozymes was indicated by LysoTracker (red). The images were obtained by confocal microscopy with a 10× objective. ( e ) The inhibitors induced the changes in F-actin polymerization. Alexa-Fluor 568-labeled phalloidin was used to visualize the F-actin of S. aureus -infected RAW264.7 cells in the absence or presence of inhibitors, which was observed by confocal microscopy (63×). White arrow = filopodia. ( f ) Treatment with PI3K inhibitors significantly increased S. aureus survival. S. aureus -infected RAW264.7 cells were treated with or without the indicated reagent. At the start of infection (0 h), the cell-associated colony-forming units (CFUs) were similar between different treatments. Bacterial survival CFUs were grown and counted at the end of infection (3 h). The data are shown as the means ± SD of three individual experiments. Significance was analyzed by one-way ANOVA followed by the Bonferroni test ( p < 0.05). * Significant difference compared with the S. aureus -infected control.

Article Snippet: PD98059, a specific inhibitor of mitogen-activated protein kinase, was purchased from TargetMol (Boston, MA, USA).

Techniques: Activation Assay, Infection, Western Blot, Positive Control, Staining, Confocal Microscopy, Labeling

Journal: Frontiers in Immunology

Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

doi: 10.3389/fimmu.2018.01517

Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

Article Snippet: To assess PAR-induced cell signaling, starved cells were treated with 0–20 µM of either PAR-1, PAR-2, or PAR-4 antagonists (Peptides International, Louisville, KY, USA) for 30 min before stimulation with FVIIa + FX or FVIIa + FX + FII at indicated doses; or cells were stimulated with 0–100 µM of PAR-1, PAR-2, or PAR-4 agonist (Peptides International); or cells were treated first with or without 50 µM mitogen-activated protein kinase inhibitor PD98059, 10 µM p38-MAPK inhibitor SB203580, 20 µM NF-kB inhibitor SN50, and 1 µM of the S6K1 inhibitor (All from Merck Millipore, Hertfordshire, UK) for 30 min and then stimulated with 10 µM of PAR-1, PAR-2, or PAR-4 agonist.

Techniques: Coagulation, Isolation, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY